The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Researchers have been developing methods for cloning mammalian animals over the past two decades. Some reported methods include the steps of (1) isolating a cell, most often an embryonic cell; (2) inserting that cell or a nucleus isolated from the cell into an enucleated oocyte (e.g., the nucleus of the oocyte was previously extracted), and (3) allowing the embryo to mature in vivo.
The first successful nuclear transfer experiment using mammalian cells was reported in 1983, where pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in live offspring(s). McGrath & Solter, 1983, Science 220:1300-1302. Subsequently, others described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs). These cells are and can give rise to pluripotent cells. Matsui et al., 1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38. In 1998, researchers reported that murine cumulus cells can be used as nuclear donors in cloning techniques for establishing cloned murine animals. Wakayama et al., 1998, Nature 394: 369-374.
Another nuclear transfer experiment was reported in 1986, where an ovine (sheep) embryonic cell was used as a nuclear donor in a cloning process that resulted in a cloned lamb. Willadsen, 1986, Nature 320:63-65. More recently, other lambs were reported to be cloned from ovine embryonic cells; serum deprived somatic cells; cells isolated from embryonic discs; and somatic mammary tissue. Campbell et al., 1996, Nature 380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385:810-813; and PCT Publications WO 96/07732 and WO 97/07669. Other approaches for cloning ovine animals involved manipulating the activation state of an in vivo matured oocyte after nuclear transfer. PCT Publication WO 97/07668. Publications that disclose cloned lambs report a cloning efficiency that is, at best, approximately 0.4%. Cloning efficiency, as calculated for the previous estimate, is a ratio equal to the number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs.
Yet another nuclear transfer experiment resulted in a cloned bovine animal (cattle), where the animal was cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor. This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U.S. Pat. No. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure. Sims & First, 1993, Theriogenology 39:313 and Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268. In addition, another publication reported that cloned bovine embryos were prepared by nuclear transfer techniques that utilized a PGC isolated from fetal tissue as a nuclear donor. Delhaise et al., 1995, Reprod Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod Dev. 37:413-424; and PCT application WO 95/10599 entitled "Embryonic Stem Cell-Like Cells."
With regard to porcine animals (swine), researchers have reported methods for obtaining chimeric animals, specifically, where a nuclear donor is placed inside an enucleated embryonic cell. Prather et al., 1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Reproduction 58: 1321-1329; and WO 94/26884, "Embryonic Stem Cells for Making Chimeric and Transgenic Ungulates," Wheeler, published Nov. 24, 1994.
Also, researchers have reported nuclear transfer experiments for porcine nuclear donors and porcine oocytes. See., e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; and Terlouw et al., 1992, Theriogenology 37: 309.
In addition, researchers have reported methods for activating porcine oocytes. Grocholova et al., 1997, J. Exp. Zoology 277: 49-56; Schoenbeck et al., 1993, Theriogenology 40: 257-266; Prather et al., 1991, Molecular Reproduction and Development 28: 405-409; Jolliff & Prather, 1997, Biol. Reprod. 56: 544-548; Mattioli et al., 1991, Molecular Reproduction and Development 30: 109-125; Terlouw et al., 1992, Theriogenology 37. 309; Prochazka et al., 1992, J. Reprod. Fert. 96: 725-734; Funahashi et al., 1993, Molecular Reproduction and Development 36: 361-367; Prather et al., Bio. Rep. Vol. 50 Sup 1. 282; Nussbaum et al., 1995, Molecular Reproduction and Development 41: 70-75; Funahashi et al., 1995, Zygote 3: 273-281; Wang et al., 1997, Biology of Reproduction 56: 1376-1382; Piedrahita et al., 1989, Biology of Reproduction 58: 1321-1329; Machaty et al., 1997, Biology of Reproduction 57: 85-91; and Machaty et al., 1995, Biology of Reproduction 52: 753-758.
There remains a long felt need for materials and methods that yield efficient nuclear transfer using a porcine nuclear donor. This long felt need is based in part upon a potential medical application, known as xenotransplantation, which includes procedures for extracting organs from porcine animals and transplanting these organs into humans in need of such organs. U.S. Pat. No. 5,589,582, Hawley et al., issued Dec. 31, 1991; PCT application WO 95/28412, Baetsher et al., published Oct. 26, 1995; PCT application WO 96/06165, Sachs et al., published Feb. 29, 1996; PCT application WO 93/16729, Bazin, published Sep. 2, 1993; PCT application WO 97/12035, Diamond et al., published Apr. 3, 1997; PCT application WO 98/16630, Piedrahita & Bazer, published Apr. 23, 1998.